Báo cáo khoa học: The C-terminus of viral vascular endothelial growth factor-E partially blocks binding to VEGF receptor-1 docx

The C-terminus of viral vascular endothelial growth factor-E partially blocks binding to VEGF receptor-1 Marie K Inder, Lyn M Wise, Stephen B Fleming and Andrew A Mercer Department of Microbiology and Immunology, University of Otago, Dunedin, New Zealand Keywords Orf virus; Parapoxvirus; vascular endothelial growth factor; VEGF-E; vascular endothelial growth factor receptor Correspondence L M Wise, Virus Research Unit, Department of Microbiology and Immunology, University of Otago, PO Box 56, Dunedin, New Zealand Fax: +64 479 7744 Tel: +64 479 7723 E-mail: lyn.wise@stonebow.otago.ac.nz (Received 12 August 2007, revised 24 October 2007, accepted 12 November 2007) Vascular endothelial growth factor (VEGF) family members play important roles in embryonic development and angiogenesis during wound healing and in pathological conditions such as tumor formation Parapoxviruses express a new member of the VEGF family which is a functional mitogen that specifically activates VEGF receptor (VEGFR)-2 but not VEGFR-1 In this study, we show that deletion from the viral VEGF of a unique C-terminal region increases both VEGFR-1 binding and VEGFR1-mediated monocyte migration Enzymatic removal of O-linked glycosylation from the C-terminus also increased VEGFR-1 binding and migration of THP-1 monocytes indicating that both the C-terminal residues and O-linked sugars contribute to blocking viral VEGF binding to VEGFR-1 The data suggest that conservation of the C-terminal residues throughout the viral VEGF subfamily may represent a means of reducing the immunostimulatory activities associated with VEGFR-1 activation while maintaining the ability to induce angiogenesis via VEGFR-2 doi:10.1111/j.1742-4658.2007.06189.x Members of the vascular endothelial growth factor (VEGF) family of molecules have emerged as major regulators of new blood vessel formation during vasculogenesis and angiogenesis [1–3] These proteins have critical roles during embryogenesis and in normal adult tissues, during wound healing and in pathological conditions such as tumor formation Currently, the mammalian VEGF family includes VEGF-A, VEGF-B, VEGF-C, VEGF-D and placenta growth factor VEGF family members exert their biological activity via a family of tyrosine kinase receptors, VEGF receptor (VEGFR)-1, VEGFR-2 and VEGFR-3 [4–6] VEGFR-1 is bound by VEGF-A, VEGF-B and placenta growth factor and is primarily expressed on endothelial and hematopoietic cells and may have a role in monocyte recruitment and pro-inflammatory gene expression VEGFR-2 is bound by VEGF-A, VEGF-C and VEGF-D and is the primary signaling receptor of VEGF-induced endothelial cell mitogenesis, angiogenesis and vascular permeability VEGFR-3 regulates formation of the lymphatic vasculature via VEGF-C and VEGF-D We, and others [7–16], have characterized a group of viral-derived VEGFs, collectively designated VEGF-E, which are encoded by members of the genus Parapoxvirus, namely Orf virus (ORFV), Pseudocowpoxvirus (PCPV), Parapoxvirus of red deer in New Zealand (PVNZ) and bovine papular stomatitis virus (BPSV) These viruses infect specific ungulates and can readily infect humans [17,18], and the resulting lesions in the skin demonstrate extensive vascular dilation, dermal edema and endothelial cell proliferation [17,19–21] Viral VEGF proteins differ from mammalian VEGF family members in that they specifically bind and activate VEGFR-2 and in general show little or no affinity for VEGFR-1 [8,9,11–16] The absence of VEGFR-1 binding is surprising given that the structural predictions for viral VEGFs are very similar to that of Abbreviations BPSV, bovine papular stomatitis virus; ORFV, Orf virus; PCPV, Pseudocowpoxvirus; PVNZ, Parapoxvirus of red deer in New Zealand; SA-HRP, streptavidin-peroxidase; VEGF, vascular endothelial growth factor; VEGFR, VEGF receptor FEBS Journal 275 (2008) 207–217 ª 2007 The Authors Journal compilation ª 2007 FEBS 207 Role of viral VEGF’s C-terminus in VEGFR binding M K Inder et al VEGF-A [10,22], and that viral VEGFs conserve many residues shown to be vital for VEGF-A binding to VEGFR-1 [23,24] Although domain-exchange studies with a viral VEGF identified loop regions that are essential for VEGFR-2 binding [25], a structural basis for the inability of viral VEGF to bind VEGFR-1 has not yet been determined There are, however, a number of structural features that may influence the specificity of viral VEGFs, including a C-terminal Pro ⁄ Thr-rich sequence, encoding potential O-linked glycosylation sites, that is unique to and highly conserved among the viral VEGFs [8,10,13–16] In this study, we examined the role of the C-terminal region of the VEGF encoded by ORFV strain NZ2 (ORFVNZ2VEGF) in the receptor-binding specificity and biological activities of the viral VEGFs Results Amino acid sequence comparisons Comparison of the predicted amino acid sequences of the viral VEGFs from ORFV strains NZ2 (ORFVNZ2VEGF) and NZ7 (ORFVNZ7VEGF), BPSV strain V660 (BPSVV660VEGF), PCPV strain VR634 and PVNZ strain RD86 (PCPVVR634VEGF) (PVNZRD86VEGF) revealed a Thr ⁄ Pro rich C-terminus, containing putative O-linked glycosylation sites (Fig 1A) that is not found in VEGF-A or other VEGF family members [10] To examine the role of this conserved C-terminus in the unique receptor specificity and biological activities of viral VEGFs, we constructed a mutant of ORFVNZ2VEGF in which the 16 C-terminal residues were deleted, designated ORFVNZ2VEGF-DC (Fig 1B) We also constructed a mutant in which the heparin-binding domain of VEGF-A was replaced with the 16 C-terminal residues of ORFVNZ2VEGF, designated VEGF-A-NZ2C (Fig 1B) Production and glycosylation state of VEGF mutants VEGF-A-NZ2C and ORFVNZ2VEGF-DC were expressed with a FLAG octapeptide at the C-terminus and then purified SDS ⁄ PAGE under reducing conditions followed by silver staining revealed bands at  26–28 and 19–20 kDa, for VEGF-A-NZ2C and ORFVNZ2VEGF-DC, respectively (Fig 2) Deglycosylation treatment with N-glycosidase reduced the monomeric size of VEGF-A-NZ2C and ORFVNZ2VEGF-DC by 2–4 kDa (Fig 2), which was similar to that seen for VEGF-A and ORFVNZ2VEGF, 208 A B Fig Sequence comparison of the parapoxviral VEGF C-termini and schematic representation of ORFVNZ2VEGF, VEGF-A and mutants (A) The C-terminal amino acid sequences of ORFVNZ2VEGF, BPSVV660VEGF, PCPVVR364VEGF, ORFVNZ7VEGF and PVNZRD86VEGF are shown [8,10,13,14] Predicted O-linked glycosylation sites are shaded gray [37] (B) Schematic representation of ORFVNZ2VEGF, VEGF-A and mutants ORFVNZ2VEGF-DC is a mutant of ORFVNZ2VEGF in which the 16 C-terminal (C-term) amino acids have been deleted VEGF-A-NZ2C is a mutant of VEGF-A in which the heparin-binding domain (HBD) has been replaced with the 16 C-terminal amino acids from ORFVNZ2VEGF All proteins are FLAG (F)-tagged at the C-terminus for detection and purification The locations of the conserved N-linked glycosylation site (N) and the variable loop regions (L1–3) are indicated by dashes and light gray boxes, respectively indicating that each contained N-linked glycosylation Further treatment with sialidase and O-glycosidase reduced VEGF-A-NZ2C and ORFVNZ2VEGF by another 2–4 kDa, although no size shift was observed for VEGF-A and ORFVNZ2VEGF-DC (Fig 2) The absence of O-linked glycosylation on ORFVNZ2VEGFDC supports the prediction that ORFVNZ2VEGF contains an O-linked glycosylation site in the 16 C-terminal residues (Fig 1) The observation that the addition of the 16 C-terminal residues to VEGF-ANZ2C are associated with the gain of O-linked glycosylation also supports this prediction (Fig 1) Deletion of the C-terminus of ORFVNZ2VEGF increases its affinity for VEGFR-1 and VEGFR-2 To investigate the role of the ORFVNZ2VEGF C-terminus in receptor specificity, we tested the ability of the VEGF mutants to bind immobilized dimeric Ig fusion proteins containing the extracellular domains of human VEGFR-1 or VEGFR-2 using a receptor-binding ELISA VEGF-A and VEGF-A-NZ2C demonstrated significant levels of binding to VEGFR-1 compared with FEBS Journal 275 (2008) 207–217 ª 2007 The Authors Journal compilation ª 2007 FEBS M K Inder et al Fig The C-terminus of ORFVNZ2VEGF has O-linked glycosylation Purified VEGF-A, VEGF-A-NZ2C, ORFVNZ2VEGF and ORFVNZ2VEGF-DC were analyzed before and after enzymatic removal of N- and O-linked sugars The proteins were resolved under reducing conditions following treatment with the indicated combinations of enzymes as described in Experimental procedures Proteins were visualized by western blotting using anti-FLAG Ig Molecular mass markers are indicated mock-purified protein at all of the concentrations tested (‡ 0.1 lgỈmL)1, P £ 0.05; Fig 3A) As reported previously [15,16], ORFVNZ2VEGF did not bind VEGFR-1 at any concentration tested (Fig 3A) By contrast, ORFVNZ2VEGF-DC showed significant levels of binding to VEGFR-1 from a concentration of 3.3 lgỈmL)1 (P £ 0.05; Fig 3A) VEGF-A and VEGF-A-NZ2C demonstrated significant levels of binding to VEGFR-2 compared with mock-purified protein at all of the concentrations tested (‡ 0.1 lgỈmL)1, P £ 0.05; Fig 3B) ORFVNZ2VEGF and ORFVNZ2VEGF-DC also showed significant binding to VEGFR-2 from a concentration of 0.4 lgỈmL)1 (P £ 0.05; Fig 3B) No significant differences in binding to VEGFR-2 were observed at any concentration between VEGF-A and VEGF-A-NZ2C or ORFVNZ2VEGF and ORFVNZ2VEGF-DC (P £ 0.05; Fig 3B) To further examine the receptor specificity of the VEGF mutants, we tested their ability to inhibit VEGF-A bind the dimerized Ig fusion proteins containing the extracellular domains of human VEGFR-1 or VEGFR-2 under soluble binding conditions using a competitive displacement ELISA [8,13] Role of viral VEGF’s C-terminus in VEGFR binding Preincubation of soluble VEGF-A or VEGF-ANZ2C with VEGFR-1 significantly inhibited the binding of the receptor to immobilized VEGF-A at all concentrations tested (‡ 0.4 lgỈmL)1, P £ 0.05), whereas ORFVNZ2VEGF did not significantly inhibit VEGFR-1 binding to VEGF-A at any concentration tested (Fig 3C) ORFVNZ2VEGF-DC did, however, inhibit binding of VEGFR-1 to VEGF-A from a concentration of 1.1 lgỈmL)1 (P £ 0.05; Fig 3C) Preincubation of soluble VEGF-A, VEGF-A-NZ2C, ORFVNZ2VEGF or ORFVNZ2VEGF-DC significantly inhibited the binding of VEGFR-2 to immobilized VEGF-A at all concentrations tested (‡ 0.2 lgỈmL)1, P £ 0.05; Fig 3D) ORFVNZ2VEGF-DC was, however, significantly more potent than ORFVNZ2VEGF from a concentration of 0.6 lgỈmL)1, whereas no significant differences were observed between VEGF-A-NZ2C and VEGF-A at any of the concentrations tested (P £ 0.05; Fig 3D) The interactions of the VEGF mutants with VEGFR-1 and VEGFR-2 were further tested in bioassays that detect receptor binding and cross-linking at the cell surface These assays made use of BaF3 cell lines expressing chimeric receptors consisting of the extracellular domain of either human VEGFR-1 or murine VEGFR-2 and the transmembrane and cytoplasmic domains of the erythropoietin receptor [26,27] Binding and cross-linking of the chimeric receptors induce cell proliferation VEGF-A and VEGF-A-NZ2C stimulated significant proliferation of cells expressing VEGFR-1 from the lowest concentration tested (‡ 1.2 ngỈmL)1, P £ 0.05), although ORFVNZ2VEGF did not induce cellular proliferation (Fig 3E) ORFVNZ2VEGF-DC stimulated significant proliferation of cells expressing VEGFR-1 from a concentration of 11 ngỈmL)1 (P £ 0.05; Fig 3E) VEGF-A, VEGF-A-NZ2C, ORFVNZ2VEGF and ORFVNZ2VEGF-DC were each able to stimulate significant proliferation of cells expressing VEGFR-2, in the presence of heparin, from a concentration of 0.8 ngỈmL)1 (P £ 0.05; Fig 3F) ORFVNZ2VEGF-DC was, however, significantly more potent than ORFVNZ2VEGF, VEGF-A and VEGF-A-NZ2C at all concentrations tested (0.8–22 ngỈmL)1, P £ 0.05; Fig 3F) In summary, ORFVNZ2VEGF-DC showed a significant increase in VEGFR-1 binding compared with ORFVNZ2VEGF in the three different assays (Fig 3A,C,E) In addition, a consistent, but not significant, decrease was observed in VEGFR-1 binding by VEGF-A-NZ2C compared with VEGF-A No FEBS Journal 275 (2008) 207–217 ª 2007 The Authors Journal compilation ª 2007 FEBS 209 Role of viral VEGF’s C-terminus in VEGFR binding M K Inder et al consistent differences were noted in VEGFR-2 binding between VEGF-A-NZ2C and VEGF-A (Fig 3B,D,F) ORFVNZ2VEGF-DC did, however, show a small, but significant increase, in VEGFR-2 binding, compared with ORFVNZ2VEGF, in two of the three assay systems (Fig 3D,F) 210 Enzymatic removal of the O-linked glycosylation of ORFVNZ2VEGF increases its affinity for VEGFR-1 and VEGFR-2 To examine the role O-linked glycosylation might play in the receptor specificity and biological activities of FEBS Journal 275 (2008) 207–217 ª 2007 The Authors Journal compilation ª 2007 FEBS M K Inder et al Role of viral VEGF’s C-terminus in VEGFR binding Fig Deletion of the C-terminus of ORFVNZ2VEGF increases binding to VEGFR-1 and VEGFR-2 Immobilized VEGFR-1–Ig (A) or VEGFR-2– Ig (B) fusion protein was incubated for h with increasing concentrations of soluble VEGF-A, VEGF-A-NZ2C, ORFVNZ2VEGF, ORFVNZ2VEGFDC or with medium alone Bound VEGF protein was detected with horseradish peroxidase-conjugated M2 anti-FLAG Ig Values are expressed as a binding index, defined as the mean increase in absorbance ± SEM at 450 nm over the background (n = 2) and are representative of three separate experiments Soluble VEGFR-1–Ig (C) or VEGFR-2–Ig (D) fusion protein was incubated for h with increasing concentrations of VEGF-A, VEGF-A-NZ2C, ORFVNZ2VEGF and ORFVNZ2VEGF-DC or with medium alone The mixture was then added to VEGF-A-coated wells to capture free VEGFR–Ig, which was detected with a biotinylated sheep anti-human Ig SA-HRP conjugate The results are presented as the percentage of the maximal absorbance of VEGFR–Ig bound Values are expressed as mean ± SEM (n = 2) and are representative of three separate experiments The abilities of the VEGF mutants to bind and cross-link VEGFR-1 (E) or VEGFR-2 (F) were tested using specific bioassay cell lines Bioassay cells were washed and resuspended in dilutions of VEGF-A, VEGF-A-NZ2C, ORFVNZ2VEGF and ORFVNZ2VEGF-DC, or with medium alone for 48 h at 37 °C DNA synthesis was quantified by [3H]thymidine incorporation and b-counting Values were expressed as a proliferation index, defined as the mean increase in cell proliferation ± SEM over the background (n = 2) and are representative of two separate experiments An asterisk indicates a significant (P £ 0.05) difference from the background level recorded when no growth factor was added A cross indicates a significant (P £ 0.05) difference in receptor binding between ORFVNZ2VEGF-DC and the equivalent concentration of ORFVNZ2VEGF the viral VEGFs, ORFVNZ2VEGF was treated with sialidase and O-glycosidase to remove the O-linked glycosylation The enzyme-treated protein, ORFVNZ2VEGF-DOglyc was then repurified and analyzed by SDS ⁄ PAGE and western blotting which revealed bands at  38–42 and 19–20 kDa, under nonreducing and reducing conditions, respectively (Fig 4A) N-Glycosidase treatment reduced the monomeric size of ORFVNZ2VEGF-DOglyc by 2–3 kDa (Fig 4A), but further treatment with sialidase and O-glycosidase did not result in a size shift These results confirm that ORFVNZ2VEGF-DOglyc has retained its N-linked glycosylation and the majority of the O-linked glycosylation has been removed To investigate the role of that ORFVNZ2VEGF O-linked glycosylation plays in receptor specificity we tested the ability of ORFVNZ2VEGF-DOglyc to bind and cross-link VEGFR-1 and VEGFR-2 in the BaF3 bioassays Consistent with our previous results, VEGF-A and ORFVNZ2VEGF-DC were able to stimulate significant proliferation of cells expressing VEGFR-1 from 1.2 and 11 ngỈmL)1, respectively (P £ 0.05), whereas ORFVNZ2VEGF did not induce cellular proliferation (Fig 4B) ORFVNZ2VEGF-DOglyc was less potent than ORFVNZ2VEGF-DC but did stimulate significant proliferation of cells expressing VEGFR-1 at the highest concentration tested (100 ngỈmL)1, P £ 0.05; Fig 4B) VEGF-A, ORFVNZ2VEGF, ORFVNZ2VEGF-DC and ORFVNZ2VEGF-DOglyc were each able to stimulate significant proliferation of cells expressing VEGFR-2, in the presence of heparin, from a concentration of 0.3 ngỈmL)1 (P£ 0.05; Fig 4C) ORFVNZ2VEGF-DC and ORFVNZ2VEGF-DOglyc were both significantly more potent than VEGF-A and ORFVNZ2VEGF at all concentrations tested (0.3– 7.4 ngỈmL)1, P £ 0.05; Fig 4C) Removal of the C-terminal residues or O-linked glycosylation of ORFVNZ2VEGF increases its recruitment of THP-1 monocytes Previous studies have shown that mammalian VEGF family members induce monocyte chemotaxis via their interaction with VEGFR-1 [28–30] Thus, using a TranswellÒ assay (Corning Costar, Corning, NY, USA), we examined the chemotactic response of THP-1 monocytes to treatment with the VEGF mutants The human THP-1 monocytic cell line has previously been shown to respond to VEGF family members in a manner similar to human peripheral blood monocytes [29] VEGF-A and VEGF-A-NZ2C were able to induce significant migration of cells from a concentration of ngỈmL)1 (P £ 0.05), whereas ORFVNZ2VEGF did not induce cell migration (Fig 5) ORFVNZ2VEGF-DC and ORFVNZ2VEGF-DOglyc were also able to induce significant migration of cells from a concentration of 20 ngỈmL)1 (P £ 0.05; Fig 5) Migration of THP-1 monocytes induced by VEGF-A, VEGF-A-NZ2C, ORFVNZ2VEGF-DC and ORFVNZ2VEGF-DOglyc was significantly inhibited by preincubation of the cells with neutralizing antibody against VEGFR-1 (P £ 0.05) (Fig 5) The increase in monocyte migration induced by ORFVNZ2VEGF-DC, compared with ORFVNZ2VEGF, was consistent with its increase in VEGFR-1 binding in the receptor-binding assays (Figs and 4) In addition, the slight decrease in monocyte migration by VEGF-A-NZ2C, compared with VEGF-A, was consistent with its decrease in VEGFR-1 binding in the receptor-binding assays (Fig 3) By contrast, the increase in monocyte migration induced by ORFVNZ2VEGF-DOglyc, compared with ORFVNZ2VEGF, was greater than the increase in VEGFR-1 binding observed in the receptor-binding assay (Fig 4) FEBS Journal 275 (2008) 207–217 ª 2007 The Authors Journal compilation ª 2007 FEBS 211 Role of viral VEGF’s C-terminus in VEGFR binding M K Inder et al Fig Removal of the C-terminal O-linked glycosylation from ORFVNZ2VEGF increases binding to VEGFR-1 and VEGFR-2 (A) Purified ORFVNZ2VEGF and ORFVNZ2VEGF-DOglyc were analyzed before and after enzymatic removal of N- and O-linked sugars The proteins were resolved under reducing conditions following treatment with the indicated combinations of enzymes as described in Experimental procedures Proteins were visualized by silver staining Molecular mass markers are indicated The ability of ORFVNZ2VEGF-DOglyc to bind and cross-link VEGFR-1 (B) or VEGFR-2 (C) was tested using specific bioassay cell lines Bioassay cells were washed and resuspended in dilutions of VEGF-A, ORFVNZ2VEGF, ORFVNZ2VEGF-DC and ORFVNZ2VEGF-DOglyc, or with medium alone for 48 h at 37 °C DNA synthesis was quantified by [3H]thymidine incorporation and b-counting Values were expressed as a proliferation index, defined as the mean increase in cell proliferation ± SEM over the background (n = 2) and are representative of two separate experiments An asterisk indicates a significant (P £ 0.05) difference from the background level recorded when no growth factor was added A cross indicates a significant (P £ 0.05) difference in receptor binding between ORFVNZ2VEGFDC or ORFVNZ2VEGF-DOglyc and the equivalent concentration of ORFVNZ2VEGF Discussion In this study, we demonstrated a role for the highly conserved O-glycosylated C-terminus in determining the unique receptor-recognition profile and biological activities of viral VEGFs Complete removal of the C-terminus of ORFVNZ2VEGF increased its binding to VEGFR-1 In addition, enzymatic removal of the O-linked glycosylation within the C-terminal region improved the ability of ORFVNZ2VEGF to activate VEGFR-1, but not to the same level as that of the deletion mutant These results indicate that the C-terminal residues play a dominant role in preventing 212 ORFVNZ2VEGF recognition of VEGFR-1 and that the O-linked sugars associated with the C-terminus play a contributing role Interestingly, a previous study using Escherichia coli-expressed viral VEGF (strain D1701), which would not have contained O-linked glycosylation, reported minimal VEGFR-1 binding [11,12] The assay employed in this study directly measures binding and cross-linking of chimeric VEGFR-1 and may be more sensitive than the endothelial cellbased assays used in the previous study Removal of the C-terminal residues from ORFVNZ2VEGF also appeared to slightly enhance signaling via VEGFR-2, as measured in the BaF3 cell proliferation assay (Fig 3F) This suggests that the C-terminal region of viral VEGF interferes with both VEGFR-1 and VEGFR-2 binding This is consistent with previous studies, which have shown that the binding sites for VEGFR-1 and VEGFR-2 overlap [23,31,32] A slight increase in VEGFR-2 binding by ORFVNZ2VEGF-DC was also seen in the competitive receptorbinding ELISA (Fig 3D) However, no difference in VEGFR-2 binding was observed in the direct receptorbinding ELISA (Fig 3B) This latter assay relies on immunodetection of the FLAG peptide fused to the VEGF protein Because the precise location of the FLAG peptide, in relation to the VEGF homology domain, varies between proteins, the accessibility of the FLAG peptide within the VEGF ⁄ VEGFR complex may also differ This may influence the sensitivity of the assay and mask the slight differences in VEGFR-2 observed in other assays Interestingly, removal of the FEBS Journal 275 (2008) 207–217 ª 2007 The Authors Journal compilation ª 2007 FEBS M K Inder et al Role of viral VEGF’s C-terminus in VEGFR binding Fig Removal of the C-terminal residues or O-linked glycosylation increases ORFVNZ2VEGF-induced VEGFR-1-dependent chemotaxis of THP-1 monocytes THP-1 monocytes (1 · 105 cells) were added to the upper chamber of a TranswellÒ insert The indicated concentrations of VEGF-A, VEGF-A-NZ2C, ORFVNZ2VEGF, ORFVNZ2VEGF-DC and ORFVNZ2VEGF-DOglyc, or medium alone were added to the lower compartments, the inserts were incubated for h at 37 °C and migrated cells that remained attached to the insert membrane were stained and counted as described in Experimental procedures Where indicated, THP-1 monocytes were preincubated with a neutralizing antibody against VEGFR-1 for 16 h and then assayed as described Results were expressed as a migration index, defined as the mean increase in cell migration, ± SEM, over background (n = 8) and are representative of three experiments Migration indexes that were significantly above that of medium only are indicated by an asterisk (P £ 0.05) A cross indicates a significant (P £ 0.05) difference between cell migration induced in the presence and absence of antibody O-linked glycosylation from ORFVNZ2VEGF also slightly increased the response seen in the VEGFR-2 BaF3 assay, to the level seen with the deletion mutant (Fig 4C) This indicates that steric hindrance by the O-linked sugars, and not the C-terminal residues, may reduce the viral VEGF’s affinity for VEGFR-2 Recently the crystal structure of ORFVNZ2VEGF was solved revealing a high similarity to the known structures of other VEGF family members [22,25] The C-terminal region, however, appeared to be highly flexible and could not be resolved by crystallography Its location adjacent to the receptor-binding face, as illustrated in Fig 6, suggests it is well placed to influence the receptor-recognition profile of viral VEGF The C-terminal residues may therefore form a direct physical block, preventing access of the VEGFRs to the receptor-binding face Curiously, replacement of the heparin-binding domain of VEGF-A with the C-terminal residues of the viral VEGF did not significantly alter the affinity of VEGF-A for VEGFR-1 or VEGFR-2 A simple explanation for this finding would be that the C-terminal residues of the VEGF-A mutant, due to the influence of adjacent residues or other structural features of VEGF-A, are not orientated towards the receptor-binding face and are therefore unable to influence receptor recognition Alternatively, the inhibitory effects of the C-terminal residues on VEGFR binding may be dependent on their interactions with other structural features of the viral VEGF Removal of the C-terminal region from ORFVNZ2VEGF did not elevate VEGFR binding to the level seen for VEGF-A, which suggests that other structural elements play a role in determining VEGFR specificity of the viral VEGFs It has been hypothesized that the inability of viral VEGF to bind VEGFR-1 may be due to the absence of a functional groove on the receptorbinding face of the homodimer [10,22] The lack of this groove may prevent viral VEGFs effectively binding the domain 2–3 linker region of VEGFR-1 In support of this model, changing Arg46 to Iso within loop of the groove region of ORFVNZ2VEGF increased its affinity for both VEGF receptors [22] In addition, replacement of both loop and loop of ORFVNZ2VEGF with those of VEGF-A partially restored binding to VEGFR-1 [22] Neither mutation, however, completely restored VEGFR-1 binding to the levels seen for VEGF-A [22] These findings in conjunction with the results of our study suggest that the groove region of the receptor binding face and the C-terminal residues make separate but additive contributions to the inability of viral VEGF to bind VEGFR-1 It would therefore be interesting to construct a viral FEBS Journal 275 (2008) 207–217 ª 2007 The Authors Journal compilation ª 2007 FEBS 213 Role of viral VEGF’s C-terminus in VEGFR binding M K Inder et al A B Fig Structural elements of ORFVNZ2VEGF involved in VEGFR binding Ribbon representation of the structure of (A) VEGF-A:Flt1-D2 (VEGFR-1 domain 2) (PDB identifier 1QTY) [32] and (B) ORFVNZ2VEGF (PDB identifier 2GNN) [22] The VEGF dimers are shaded gray with one monomer in a darker shade, and with loops 1, and 3, shaded blue, green and red, respectively VEGFR1 domain is shaded purple Residues Ser94–Asn99 within loop of one of the ORFVNZ2VEGF monomers are missing, as they were disordered due to their intrinsic flexibility The residues of VEGF-A that form the groove implicated in VEGFR-1 binding, and the residues of ORFVNZ2VEGF in the equivalent positions, are shaded black The flexible C-terminus of each ORFVNZ2VEGF monomer is labeled and shaded orange, with the disordered residues Thr120– Arg133 drawn schematically as dashes The putative O-linked glycosylation sites within the C-termini are shaded yellow VEGF mutant in which, loops and are replaced with those from VEGF-A, and the C-terminal residues are deleted, to ascertain whether these regions are the determinants of VEGFR recognition or if additional structural features are involved The C-terminal residues are conserved among all members of the viral VEGF family Recently, a variant viral VEGF from BPSV has been reported that shows greater recognition of VEGFR-1 and VEGFR-2 than the other viral VEGFs [8] BPSVV660VEGF varies from the other viral VEGFs in its C-terminal residues, yet conserves the O-linked glycosylation sites This supports our finding that the specific amino acids within 214 the C-terminus influence the receptor-recognition profile of viral VEGFs It was also proposed that BPSVV660VEGF residues adjacent to the receptor-binding face influence the orientation of loop and the width of the groove, thereby affecting its ability to bind the VEGFRs [8] The orientation of the receptor-binding face of BPSVV660VEGF may therefore be such that the C-terminal residues have less interaction with the loop regions Alternatively, this region of the other viral VEGFs, which differ from BPSVV660VEGF, maybe act as a site of interaction with the C-terminal residues VEGFR-1 activation has been shown to mediate VEGF-induced immunostimulatory activities such as dendritic cell activation, monocyte migration, inflammatory cytokine induction and other important antiviral immune defenses [29,30,33,34] Removal of either the C-terminal residues or O-linked glycosylation from the viral VEGF resulted in an increase in VEGFR-1mediated migration of THP-1 monocytes Conservation of the C-terminal residues throughout the viral VEGF family may therefore represent a means of viral immune evasion The viral VEGFs, however, retain sufficient binding of VEGFR-2 to potently induce vascular dilation, dermal edema and proliferation of endothelial cells, thereby contributing to the proliferative and highly vascularized nature of parapoxvirus lesions [15,35,36] Experimental procedures Expression vectors Expression vectors for VEGF-A (murine VEGF isoform 164) and ORFVNZ2VEGF were derived from pAPEX-3 and have been described previously [15] A DNA fragment containing nucleotides 4–368 of ORFVNZ2VEGF was amplified by PCR from viral DNA with the following primers: NZ2-DC 5¢ (5¢-AGCGCCCGGCGCGCCAGA AGTTGCTCGTCGGCATAC-3¢, AscI site underlined) and NZ2-DC 3¢ (5¢-ACTCGAACGCGTTCGTGGTCTA CAATCGCA-3¢, MluI site underlined) A DNA fragment containing nucleotides 4–458 of VEGF-A and 55 nucleotides from the C-terminus of ORFVNZ2VEGF was amplified from pAPEX–mVEGF-A [15] with the following primers: mV-for 5¢ (5¢-CATGGCGCGCCTGATGAAC TTTCTGCTGTCTTGG-3¢) and mV-NZ2C 3¢ (5¢-CTGAC GCGTGCGGCGTCTTCTGGGCGGCCTTGTGGTCGT CGGTGGCGTGGTTGTGAACTTTGGTCTGCATTCA CATCGGCT-3¢) The PCR products were digested with AscI and MluI and ligated to pAPEX–mVEGF-A [15], from which the DNA sequence encoding VEGF-A but not the FLAG octapeptide (IBI ⁄ Kodak, Rochester, NY, USA) had been removed by digestion with AscI FEBS Journal 275 (2008) 207–217 ª 2007 The Authors Journal compilation ª 2007 FEBS M K Inder et al Protein synthesis from the expression vectors described above gave rise to secreted polypeptides tagged with the FLAG octapeptide at their C-termini The C-terminal deletion mutant of ORFVNZ2VEGF was designated ORFVNZ2VEGF-DC, and the domain exchange mutant of VEGF-A with the ORFVNZ2VEGF O-glycosylated C-terminus was designated VEGF-A-NZ2C Role of viral VEGF’s C-terminus in VEGFR binding h The mixture was then transferred to plates coated with VEGF-A and incubated at 25 °C for h to capture the unbound VEGFR–Ig fusion protein The captured VEGFR–Ig fusion protein was detected by biotinylated anti-human Ig (Dako, Glostrup, Denmark) and streptavidin-peroxidase (SA-HRP; Sigma) and tetramethylbenzidine substrate reagent and quantified by measuring the absorbance at 450 nm Recombinant protein production Recombinant FLAG-tagged proteins were expressed in 293-EBNA cells, purified and quantitated as previously described [15] A mock elution sample was obtained from conditioned medium of pAPEX-3-transfected 293-EBNA cells that underwent the same purification process Bioassays for the binding and cross-linking of the extracellular domains of VEGFR-1 and VEGFR-2 Proteins were treated with N-glycosidase F (Roche, Mannheim, Germany), sialidase (neuraminidase, Roche), and O-glycosidase (Roche), and resolved by SDS ⁄ PAGE and visualized by silver staining or western blotting, as previously described [14] Bioassays for monitoring the binding and cross-linking of VEGFR-1 and VEGFR-2, using BaF3-derived cell lines expressing chimeric receptors consisting of the extracellular, ligand-binding domains of human VEGFR-1 or mouse VEGFR-2 and the transmembrane and cytoplasmic domains of the erythropoietin receptor, were carried out as previously described [26,27] Briefly, the bioassay cell lines were incubated with various concentrations of purified growth factors for 48 h at 37 °C DNA synthesis was quantified by measuring [3H]thymidine incorporation during a further 16 h incubation ELISA receptor-binding assay Chemotaxis assay Maxisorp 96-well immunoplates (Nunc, Roskilde, Denmark) were coated with 500 ngỈmL)1 VEGFR-1–Ig or VEGFR-2–Ig fusion proteins (R&D Systems, Minneapolis, MN, USA) in coating buffer (15 mm Na2CO3, 35 mm NaHCO3, pH 9.6) at °C for 16 h and blocked with 0.5% BSA and 0.02% Tween 20 at room temperature for h Plates were washed between steps with wash buffer (NaCl ⁄ Pi and 0.02% Tween 20) Immobilized VEGFR–Igs were then incubated with a titration of purified VEGFs at room temperature for h Captured VEGF was detected by horseradish peroxidase-conjugated M2 anti-FLAG Ig (Sigma, St Louis, MO, USA) and developed with tetramethylbenzidine substrate reagent (B&D Biosciences, San Diego, CA, USA) and quantified by measuring absorbance at 450 nm Chemotaxis assays using THP-1 monocytes were carried out in 24-well plates containing TranswellÒ inserts of lm pore size (Corning Costar, Corning, NY, USA), as previously described [8] Briefly, monocytes were loaded into inserts with various concentrations of purified growth factor in the bottom compartment and then incubated for h at 37 °C Nonmigrated cells were removed from the upper side of the filter membrane and the adherent cells on the lower side were fixed in gluteraldehyde then stained using Gill’s hemotoxylin For a quantitative assessment of migrated cells, a total of four fields of ·40 magnification from two different wells was counted Protein deglycosylation ELISA competitive displacement receptor binding assay Maxisorp 96-well immunoplates were incubated with 400 ngỈmL)1 VEGF-A in coating buffer at °C for 16 h and blocked with 1% BSA and 0.02% Tween 20 at 37 °C for 45 Plates were washed between steps with wash buffer Samples of purified growth factors, serially diluted in binding buffer (NaCl ⁄ Pi with 0.4% BSA, 0.02% Tween 20 and lgỈmL)1 heparin in VEGFR-2 assays only), were incubated with 300 ngỈmL)1 human VEGFR-1– Ig or VEGFR-2–Ig in non-absorbent plates at 25 °C for Enzymatic removal of O-linked glycosylation from ORFVNZ2VEGF Purified protein (150 lg) was diluted in 0.05 m sodium phosphate buffer (pH 7) containing 0.1% SDS Twenty mU of sialidase and 25 mU of O-glycosidase was added and the mixture was incubated at 37 °C for h The protein was then re-purified and quantitated, as previously described [15] Statistical analysis Statistical analysis was performed using analysis of variance (single factor anova) with significant points of difference (P £ 0.05) determined using Tukey’s test FEBS Journal 275 (2008) 207–217 ª 2007 The Authors Journal compilation ª 2007 FEBS 215 Role of viral VEGF’s C-terminus in VEGFR binding M K Inder et al Acknowledgements This study was partially supported by the Health Research Council of New Zealand Lyn Wise was supported in part by the University of Otago Health Sciences Career Development Program Postdoctoral Fellowship Award We thank Nicola Real for expert technical assistance References Carmeliet P (2005) VEGF as a key mediator of angiogenesis 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VEGFR specificity of the viral VEGFs It has been hypothesized that the inability of viral VEGF to bind VEGFR-1 may be due to the absence of a functional groove on the receptorbinding face of the. .. Deletion of the C-terminus of ORFVNZ 2VEGF increases its affinity for VEGFR-1 and VEGFR-2 To investigate the role of the ORFVNZ 2VEGF C-terminus in receptor specificity, we tested the ability of the VEGF. .. [8,13] Role of viral VEGF? ??s C-terminus in VEGFR binding Preincubation of soluble VEGF- A or VEGF- ANZ2C with VEGFR-1 significantly inhibited the binding of the receptor to immobilized VEGF- A at all